Proteins carry out virtually all of the complex processes of life, from photosynthesis to signal transduction and the immune response. To understand these intricate activities, it would be useful to learn how proteins interact with other molecules to perform their functions. Polypeptide library screening techniques are extremely valuable tools in investigating and manipulating these molecular interactions. In general, screening a polypeptide library relies on the expression of a plurality of polypeptide variants followed by the isolation and amplification of those variants that exhibit a function of interest, e.g., variants that bind to a particular ligand. However, isolating such variants can be critically dependent upon maintaining library diversity.
Differential expression rates among the polypeptide variants in a library can be detrimental to the maintenance of library diversity, and, therefore, to the selection of functional sequences. For example, certain highly expressed variants can become enriched in a population following repeated cycles screening, isolation, and amplification not because they meet the screening criterion, but because they are over-represented due solely to growth advantages. As a consequence, desirable variants that are expressed at lower levels can become underrepresented or can even become lost. This is particularly true of polypeptide variants comprising unnatural amino acids due to an inherent in vivo expression bias for proteins containing only natural amino acids.
What is needed in the art are new strategies for controlling the expression levels throughout a population of expressed polypeptide variants, especially populations that include variants comprising unnatural amino acids, while avoiding adverse effects on the diversity of variants in the population. The invention described herein fulfills these and other needs, as will be apparent upon review of the following disclosure.